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The present invention has for an object a koji mold which expresses at
least 2 times more endo- and exo-peptidases than the wild type strain of
Aspergillus oryzae CNCM I-1882, and especially at least 30 mU of
endopeptidase activity, at least 30 mU of leucine-amino-peptidase and at
least 10 mU of prolyldipeptidyl-peptidase activity per ml of supernatant
when grown in a minimal medium containing 0.2% soy bean proteins. The
invention also provides a DNA-binding protein of Aspergillus oryzae (AREA)
having at least the amino-acid sequence from amino-acid 1 to amino-acid
731 of SEQ ID NO:2 or functional derivatives thereof. The invention also
provides a DNA molecule that comprises an areA gene encoding the
DNA-binding protein according to the invention. In a fourth aspect, the
invention provides a method for over-producing proteolytic enzymes,
comprising cultivating a koji mold according to the invention in a
suitable growth medium under conditions where the mold expresses enzymes,
and optionally isolating the enzymes in the form of a concentrate. In
another aspect, the invention provides the a method for hydrolyzing
protein-containing materials using the koji mold of the invention. In a
last further aspect, the invention provides a food product comprising a
protein hydrolysate obtainable by fermentation with a koji mold of the
invention of a material comprising proteins and at least 5 mM of
L-glutamine.

1. An isolated koji mold which expresses at least 2 times more endo- and exo-peptidases than the wild type strain Aspergillus oryzae CNCM I-1882.

2. An isolated koji mold according to claim 1, which expresses at least 30 mU of endopeptidase activity, at least 30 mU of leucine-amino-peptidase activity and at least 10 mU of proline-dipeptidyl-peptidase activity per ml of supernatant when
the koji mold is grown in a minimal medium comprising 0.2% soy bean proteins.

3. An isolated koji mold according to claim 1, which expresses the recited proteolytic activities in the presence of at least 5 mM L-glutamine.

4. An isolated koji mold according to claim 1, further comprising an areA gene, which is not repressed when the koji mold is grown in a minimal medium comprising repressive amounts of L-glutamine.

5. An isolated koji mold according to claim 4, further comprising a truncated areA gene which encodes a C-terminally-truncated AREA protein, wherein the C-terminally-truncated AREA protein remains functional, and wherein the
C-terminally-truncated AREA protein is not substantially repressed when the koji mold is grown in a minimal medium comprising repressive amounts of L-glutamine.

6. An isolated koji mold according to claim 4, further comprising multiple copies of an integrated areA gene.

7. An isolated koji mold according to claim 5, wherein the truncated areA gene is operably linked to at least one regulatory sequence which directs

over-expression of the areA gene.

8. A koji mold according to claim 5, wherein the areA gene has the nucleotide sequence defined by nucleotides 1189-1604 and 1704-3480 of SEQ ID NO:1 or functional derivatives thereof which comprise nucleotide sequences encoding substantially
identical amino acid sequences.

9. An isolated koji mold according to claim 1 wherein the koii mold is selected from the group consisting of Aspergillus, Rhizopus and Mucor.

11. A method for over-producing proteolytic enzymes, comprising the step of cultivating a koji mold according to claim 1 in a suitable growth medium under conditions where the mold expresses proteolytic enzymes.

12. The method of claim 11, further comprising the step of isolating the proteolytic enzymes in the form of a concentrate.

Hydrolyzed proteins, which are widely used in the food industry, may be prepared by hydrolysis of protein material with acid, alkali or enzymes. Various methods have been used koji molds for the preparation food products, which are hydrolyzed by
action of a large variety of secreted amylases, proteinases and peptidases. Koji molds are those traditionally used for making a koji culture (U.S. Pat. No. 4,308,284) including cells of the genus Aspergillus, Rhizopus and/or Mucor, especially
Aspergillus soyae, Aspergillus oryzae, Aspergillus phoenicis, Aspergillus niger, Aspergillus awamori, Rhizopus oryzae, Rhizopus oligosporus, Rhizopus japonicus, Rhizopus formosaensis, Mucor circinelloides, Mucor japanicus, Penicillium glaucum and
Penicillium fuscum, for example.

According to the rules of the International Code of Botanical Nomenclature (ICBN), Aspergillus is an anamorphic genus. This means that true Aspergillus only reproduce asexually through conidiophores. However, the typical Aspergillus
conidiophore morphology can also be found in fungi that can reproduce sexually via ascospores. Some Aspergillus taxonomists caused confusion, because they did not adhere to ICBN terminology. Instead, they attempted to make various revisions of
taxonomical schemes to include Aspergillus nidulans in this genus, despite the fact that its taxonomically correct name is Emericelia nidulans (Samson, In: Aspergillus. Biology and Industrial Applications, pp 355-390, Ed. by Bennett and Klich, Boston)

EP417481 (Societe des Produits Nestle) thus describes a process for the production of a fermented soya sauce, in which a koji is prepared by mixing a koji culture with a mixture of cooked soya and roasted wheat, the koji is then hydrolyzed in
aqueous suspension for 3 to 8 hours at 45.degree. C. to 60.degree. C. with the enzymes produced during fermentation of the koji culture, a moromi is further prepared by adding sodium chloride to the hydrolyzed koji suspension, the moromi is left to
ferment and is then pressed and the liquor obtained is pasteurized and clarified.

EP429760 (Societe des Produits Nestle) describes a process for the production of a flavoring agent in which an aqueous suspension of a protein-rich material is prepared, the proteins are solubilized by hydrolysis of the suspension with a protease
at pH6.0 to 11.0, the suspension is heat-treated at pH 4.6 to 6.5, and the suspension is ripened with enzymes of a koji culture.

Likewise, EP96201923.8 (Societe des Produits Nestle) describes a process for the production of a meat flavor, in which a mixture containing a vegetal proteinaceous source and a vegetal carbohydrates containing source is prepared, said mixture
having initially at least 45% dry matter, the mixture is inoculated with a koji culture and by one or more another species of microorganisms involved in the traditional fermentation of meat, and the mixture is incubated until meat flavors are formed.

However, on the one hand, acid or alkaline hydrolysis can destroy the essential amino acids produced during hydrolysis thus reducing the nutritional value, whereas enzymatic hydrolysis rarely goes to completion so that the hydrolyzed protein
contains substantial amounts of peptides. The optimization and further development of koji processes have been seriously hampered by the lack of knowledge on the nature of the hydrolytic enzymes, their regulation and how process parameters affect their
expression and activity (e.g. temperature, pH, water activity, and salt concentration).

The areA gene encodes a positively acting DNA-binding protein (AREA), belonging to the GATA family of transcription factors, that is required for the utilization of all nitrogen sources except ammonia or L-glutamine.

Under nitrogen de-repressed conditions, signaled by high intracellular levels of glutamine, areA expression is down regulated by three mechanisms: 1) the AREA protein is inactivated, 2) areA transcription is halted and 3) by action of the 3'
untranslated trailer sequence (3'-UTS) areA mRNA degradation is enhanced (Platt et al., EMBO J., 15, 2791-2801, 1996). In the absence of a functional AREA protein, only ammonia or L-glutamine can be utilized as nitrogen source. Consequently,
loss-of-function areA mutants can utilize only ammonia or L-glutamine as nitrogen sources (Arst et al., 1973).

Observations in koji fermentation suggest that nitrogen metabolite repression is a major parameter in koji fermentation. For instance, high levels of L-glutamine are shown to negatively affect proteolytic activity in koji fermentation.

Furthermore, it has been observed that high levels of proteolytic activity and glutaminase activity are two mutually exclusive conditions in koji fermentation (Ushijima et al., Agric. Biol. Chem., 51, 1051-1057, 1997). For instance, addition
of 25 mM L-glutamine into a minimal growth medium containing 0.1% wheat gluten reduces endoproteolytic enzyme activity about 40-50 fold. This phenomenon may be explained by postulating that L-glutamine is necessary for the induction of glutaminase.
However, since L-glutamine is also the effector of nitrogen metabolite repression, the expression of proteolytic enzymes is suppressed when glutaminase is induced.

With regard to the fact that glutaminase suitably converts L-glutamine into L-glutamic acid which is an important natural taste enhancer (see WO95/31114), there is hence a need to overcome L-glutamine mediated suppression of proteolytic enzymes,
allowing simultaneous expression of glutaminase and proteolytic enzymes in koji molds.

In addition, depending on the nature of the protein and the enzymes used for proteolysis, the peptides formed can however have extremely bitter tastes and are thus organoleptically undesirable. There is hence also a need for methods of
hydrolyzing proteins leading to high degree of protein hydrolysis and to hydrolysates with excellent organoleptic properties.

Finally, biochemical analysis of residual peptides in cereals hydrolyzed by koji molds, e.g. wheat gluten, shows that a considerable amount of L-glutamine remains sequestered in proline containing peptides (Adler-Nissen, In: Enzymatic hydrolysis
of food proteins. Elsevier Applied Sciences Publishers LTD, p120, 1986). There is hence also a need for methods of hydrolyzing proteins leading to liberation of high amount of L-glutamine.

SUMMARY OF THE INVENTION

The present invention has for object a koji mold which is capable to expressing at least 2 times more endo- and exo-peptidases than the wild type strain Aspergillus oryzae CNCM I-1882, and especially at least 30 mU of endopeptidase activity, at
least 30 mU of leucine-amino-peptidase activity and at least 10 mU of prolyl-dipeptidyi-peptidase activity per ml of supernatant when grown in a minimal medium containing 0.2% soy bean proteins.

In a second aspect, the invention also provides a DNA-binding protein of Aspergillus oryzae (AREA) having at least the amino-acid sequence from amino-acid 1 to amino-acid 731 of SEQ ID NO:2 or functional derivatives thereof.

In a third aspect, the invention provides a DNA molecule that comprises an areA gene encoding the DNA-binding protein according to the invention.

In a fourth aspect, the invention provides a method for over-producing proteolytic enzymes, comprising cultivating a koji mold according to the invention in a suitable growth medium under conditions that the mold expresses enzymes, and optionally
isolating the enzymes in the form of a concentrate.

In another aspect, the invention provides the use of the koji mold of the invention to hydrolyze protein-containing materials.

In a last further aspect, the invention provides a food product comprising a protein hydrolysate obtainable by fermentation with a koji mold of the invention of a material comprising proteins and at least 5 mM of L-glutamine.

DETAILED DESCRIPTION OF THE INVENTION

Within the following description, the percentages are given by weight except where otherwise stated. The amino acid or nucleotide sequences referred as "SEQ ID NO:" are always presented in the sequence listing hereafter. One
leucine-aminopeptidase enzyme unit is defined as the amount of enzyme which produces 1 .mu.mol p-nitroaniline per minute at 37.degree. C. from the substrate leucine-p-nitroanilide (absorption measured at 400 nm; .epsilon.=10,500 M.sup.-1 cm-.sup.-1).
One prolyl-dipeptidyl-peptidase enzyme unit is defined as the amount of enzyme which produces 1 .mu.mol p-nitroaniline per minute at 37.degree. C. from the substrate Alanine-Proline-p-nitroanilide (absorption measured at 400 nm; .epsilon.=10,500
M.sup.-1 cm.sup.-1). One endopeptidase enzyme unit is defined as the amount of enzymes which produces 1 .mu.mol of TCA-soluble peptides per minute at 37.degree. C. from the resorufin-labeled casein substrate under prescribed conditions (Boehringer Cat
No. 1080733; absorption measured at 574 nm).

The term "koji" is defined as the product of the fermentation with a koji mold culture of a mixture of a source of proteins and a source of carbohydrates, especially of a mixture of a leguminous plant or of a cooked oleaginous plant and of a
cooked or roasted cereal source, for example of a mixture of soya or cooked beans and of cooked or roasted wheat or rice.

Likewise, the expression "functional derivative of an enzyme" includes all amino acid sequences which differ by substitution, deletion, addition of some amino acids, for instance 1-20 amino acids, but which keep their original activities or
functions. The selection of a functional derivative is considered to be obvious to one skilled in the art, since one may easily creates variants of the truncated AREA protein (see SEQ ID NO:2) by slightly adapting methods known to one skilled in the
art, for instance the methods described by Adams et al. (EP402450; Genencor), by Dunn et al. (Protein Engineering, 2, 283-291, 1988), by Greener et al (Strategies, 7, 32-34, 1994), and/or by Deng et al. (Anal. Biochem, 200, 81, 1992).

In particular, a protein may be generally considered as a derivative of another protein, if its sequence is at least 85% identical to the protein, preferably at least 90%, in particular 99%. In the context of the present disclosure, the identity
is determined by the ratio between the number of amino acids of a derivative sequence which are identical to those of the truncated AREA protein (see SEQ ID NO:2) and the total number of or amino acids of the derivative sequence.

The present invention thus concerns any koji molds providing an enhanced expression of proteolytic enzymes, leading to high degree of protein hydrolysis and to hydrolysates with excellent organoleptic properties. Accordingly, these koji molds
express (1) high levels of endopeptidases such as those capable to produce TCA-soluble peptides at 37.degree. C. from casein, and (2) high levels of exo-peptidases such as the leucine-amino-peptidase that eliminates N-terminal leucines (Deng et al.,
Anal. Biochem., 200, 81, 1992) and the prolyl-dipeptidyl-peptidase which eliminates N-terminal X-Proline dipeptides, wherein X may be any amino-acid (Barrett et al., In Mammalian Proteases: A Glossary and Bibliography, N.Y., Acad. Press, 2, p.132,
1986).

With regard to the fact that koji molds of the invention provide an enhanced prolyl-dipeptidyl-peptidase activity, they may suitably be used for liberating L-glutamine remains sequestered in proline containing peptides.

Koji molds providing the following enhanced expression of proteolytic enzymes are particularly adapted for the purpose of the invention: at least about 30 mU/ml*, preferably at least about 50 mU/ml* of endopeptidase activity; at least about 30
mU/ml*, preferably at least about 50 mU/ml* of leucine-amino-peptidase activity; and at least 10 mU/ml*, preferably at least about 15 mU/ml* of proline-dipeptidyl-peptidase activity (* per ml of supernatant when grown in a minimal medium containing 0.2%
soy bean proteins).

In addition, koji molds that overcome L-glutamine mediated suppression of proteolytic enzymes, allowing simultaneous expression of glutaminase and proteolytic enzymes, are also part of the invention. These koji molds thus may express the
above-mentioned proteolytic activities when grown in a minimal medium containing 0.2% soy bean proteins and at least 5 mM L-glutamine (0.073% w/w), for instance.

Koji molds of the invention may be obtained by random UV and/or chemical mutagenesis, followed by selection of mutagenized koji mold providing the required phenotypic characteristics.

Selection of mutagenized koji mold particularly containing a mutagenized areA gene which is not repressed, when the mutagenized mold is grown in a minimal medium containing repressive amounts of L-glutamine, suitably achieved the needs of the
present invention. To this end, areA mutants may be easily selected by classical random mutagenesis (V, chemical) and selection on plates containing about 100 mM methyl ammonium chloride and 0.2% soy protein, for example.

It has to be noted that the prolyl-dipeptidyl-peptidase activity that is not naturally controlled by the areA gene expression, is enhanced against all expectations when the areA gene is de-repressed. Since expression of the
prolyl-dipeptidyl-peptidase activity is induced by peptides (unpublished results), this AREA-dependent increase in activity may in fact be caused by the enhanced liberation of peptides by the endoproteases that are under areA control.

With regard to the fact that random UV and/or chemical mutagenesis is time consuming, it would be also more adequate to construct koji molds of the invention by recombinant technology. Accordingly, a koji mold of the invention may preferably
contain a recombinant areA gene which is truncated so as the C-terminally truncated AREA protein remains functional but not repressed when the mold is grown in a minimal medium containing repressive amounts of L-glutamine. It has to be noted that this
truncation leads also to an areA mRNA that is less sensitive to mRNA degradation.

Truncation may be effected by cutting the native areA gene to a pre-determined region, and by introducing a terminater region thus allowing transcription of a truncated areA mRNA. Truncation is preferably effected downstream of the sequence
encoding the DNA binding domain of AREA, that can be easily identified by 17 amino acid loop bound two pairs of cysteine residues. More precisely, truncation may be effected downstream of the areA sequence encoding the conservative amino-acid structure
cysteine-2X-cysteine-17X-cysteine-2X-cysteine, where in X is any amino-acids and the numbers 2 and 17 refer to the number of amino-acids (Caddick et al., Antonie van leeuwenhoek, 65, 169-177, 1994). This truncation may be particularly carried out in the
100 amino-acids following the areA sequence encoding the DNA binding domain.

Any functional fungal areA gene may be used in the context of the present invention, and in particular any functional areA gene capable of hybridizing under stringent conditions to the areA gene of Aspergillus oryzae having the nucleotide
sequence from nucleotide 1189 to nucleotide 3846 of SEQ ID NO: 1 or functional derivatives thereof due to the degeneracy of the genetic code.

A functional areA gene may be obtained in substantially purified form by using the method described within the following examples from any strain of Aspergillus oryzae. Alternatively, an areA gene may be (1) detected also from other genera or
species of fungals by use of DNA probes derived from the nucleotide sequence SEQ ID NO:1 in a stringent hybridization assay, and (2) recovered by the well known Reverse-PCR method by use of suitable primers derived from SEQ ID NO:1 encompassing the areA
gene. In a further aspect, an areA gene may also be in-vitro synthesized and then multiplied by using the polymerase chain reaction, for instance.

A suitable truncated areA gene thus may particularly consist of the nucleotide sequence defined by nucleotides 1189-1604 and 1704-3480 of SEQ ID NO:1 (SEQ ID NO:1 contains an intron) or functional derivatives thereof due to the degeneracy of the
genetic code, for example. This truncated gene thus encodes for the AREA DNA-binding protein of Aspergillus oryzae having the amino-acid sequence from amino-acid 1 to amino-acid 731 of SEQ ID NO:2, that is required for the utilization of all nitrogen
sources except ammonia or L-glutamine.

This truncated areA gene then may be introduced in a vector, e.g. a replicative plasmid or an integrative circular or linearized non replicative plasmid, and may be operably linked to regulatory sequences that regulate a different gene in the
said organism of origin or that regulate a different gene in a foreign organism (promoter and/or a terminator), for example. A regulatory sequence other than the native regulatory sequence will generally be selected for its high efficiency or desirable
characteristic, such as, in case of a promoter inducibility or high expression capacity, for example.

If heterologous expression is preferred, meaning that the gene of the invention is expressed in another organism than the original host (strain, variety, species, genus, family, order, class or division) the regulatory sequences are preferably
derived from an organism similar or equal to the expression host. For example, if the expression host is an Aspergillus, then the regulatory sequences will be derived from Aspergillus. The promoter suitable for constitutive expression, preferably in a
fungal host, may be a promoter from the following genes: glycerolaldhehyde-3-phosphate dehydrogenase, phospho-glycerate kinase, triose phosphate isomerase and acetamidase, for example. Promoter suitable for inducible expression, preferably in a fungal
host, may be a promoter from the following genes: endoxylanase IIA, glucoamylase A, cellobiosehydrolase, amylase, invertase, alcohol dehydrogenase and amyloglucosidase. The selection of a desirable regulatory sequence operably linked to a sequence of
the invention and capable of directing the expression of the said nucleotide sequence is considered to be obvious to one skilled in the art.

The vector may also comprise a selection marker to discriminate host cells into which the recombinant DNA material has been introduced from cells that do not comprise the said recombinant material. Such marker genes are, for example in case
fungal expression is preferred, the known ga-2, pyrg, pyr4, pyrA, trpC, amdS or argB genes. The DNA molecule may also comprise at least one suitable replication origin. Suitable transformation methods and suitable expression vectors provided with a
suitable transcription promoter, suitable transcription termination signals and suitable marker genes for selecting transformed cells are already known in the literature for many organisms including different Aspergillus, Rhizopus and Mucor. In the
event fungal expression is required, the expression system described in EP278355 (Novartis) may be thus particularly adapted.

Recombinant koji molds may be obtained by any method enabling a foreign DNA to be introduced into a cell. Such methods include transformation, electroporation, or any other technique known to those skilled in the art.

In the context of the present invention, koji molds are those traditionally used for making a koji culture including cells of the genus Aspergillus (ICBN taxonomy), Rhizopus and/or Mucor. Among those, the following species may be used, including
Aspergillus soyae, Aspergillus oryzae (ATCC 20386), Aspergillus phoenicis (ATCC 14332), Aspergillus niger (ATCC 1004), Aspergillus awamori (ATCC 14331), Rhizopus oryzae (ATCC 4858), Rhizopus oligosporus (ATCC 22959), Rhizopus japonicus (ATCC 8466),
Rhizopus formosaensis, Mucor circinelloides (ATCC 15242), Mucor japanicus, Penicillium glaucum and Penicillium fuscum (ATCC 10447). Strains referred by an ATCC number are accessible at the American Type Culture Collection, Rockville, Md. 20852, US.
The invention is not limited by such indications that were rather give to enable one skilled in the art to carry out the invention.

Recombinant cells of the invention may comprise the truncated areA gene stably integrated into the chromosome or on a replicative plasmid. Among all recombinant cells of the invention thus created, the present invention has particularly for
object the strains A. oryzae CNCM I-1881, CNCM I-1883

and CNCM I-1884.

Preferably, only one functional truncated areA gene is integrated into the chromosome under the control of regulatory sequences that are native to the host organism.

In order to stably integrate into the chromosome of eucaryotic cells only one functional truncated areA gene which is fused to a promoter and a terminator which are native to the host organism, the DNA molecule of the invention may be integrated
by slightly adapting the process of Ruiter-Jacobs et al. (Curr. Genet., 16, 159-163, 1989), i.e.,

(1) preparing a non-replicative DNA fragment by ligating the truncated areA gene, which is operably linked to a promoter and terminator that are native to the host organism, downstream the DNA sequence encoding an essential gene, said gene being
inactivated by at least one mutation and/or one deletion (this essential gene may be any genes involved in RNA synthesis, such as the pyrG gene in case A. oryzae is used, for example); (2) selecting a host organism containing the essential gene which is
however inactivated by another mutation(s) or deletion(s); (3) transforming said host organism with the non replicative DNA fragment; (4) identifying integrate transformants in which the DNA fragment is integrated so as to restore the native function of
the essential gene; (5) selecting an integrate transformant in which only one DNA fragment is integrated. Over-expression of the AREA DNA-binding protein may be obtained by incorporation of the truncated areA gene in an expression host, said areA gene
being operably linked to one or more regulatory sequences which serve to increase expression levels of the AREA protein of the invention.

The over-expression can be further achieved by introducing (replicative plasmid) or integrating (by integration in the genome) multiple copies of the functional truncated areA gene of the invention. As examples of koji molds containing multiple
copies of a functional truncated areA genes, the transformants Aspergillus oryzae A (see example 1), Aspergillus oryzae xprD1 (see example 3) and Aspergilus oryzae NF1 containing pNFF68 (see example 4) were deposited under the Budapest Treaty where they
respectively receive the deposit numbers CNCM I-1881, CNCM I-1883 and CNCM I-1884.

The invention is also directed to a process for over-producing proteolytic enzymes comprising, providing koji mold of the invention in a suitable growth medium under conditions that the mold expresses proteolytic enzymes, and optionally isolating
the enzymes in the form of a concentrate, for example by removing solids from the fermentation broth by centrifugation or filtration. The selection of the appropriate medium may be based on the choice of expression host and/or based on the regulatory
requirements of the DNA recombinant material. Such media are well-known to those skilled in the art. After fermentation, the molds can be removed from the fermentation broth by centrifugation or filtration.

Typical L-glutamine concentrations reached during koji hydrolysis in liquid system may be 0.5-1% w/w, for example. The present koji molds are thus particularly adapted for hydrolyzing any protein containing materials, in particular those
containing high amounts of L-glutamine (more than 5 mM). These protein containing materials may be mixtures of a source of proteins and a source of carbohydrates, especially a mixture of a leguminous plant or of a cooked oleaginous plant and of a cooked
or roasted cereal source, for example a mixture of soya or cooked beans and of cooked or roasted wheat or rice.

Compositions containing wheat gluten are particularly adapted for the purpose of the present invention, since high amounts of L-glutamine remains sequestered in proline containing peptides when wheat gluten is hydrolyzed by traditional koji
cultures.

In the event one may try, after or during hydrolysis with koji molds, to further liberate as much as possible L-glutamine linked to proline residues, the present invention provides a method in which the koji mold of the invention of the invention
is used in combination with at least an enzyme or a microorganism providing a prolidase activity, that is to say an enzyme which has a high level of specificity towards dipeptides of the X-Pro type (Ezespla et al., Ap. Env. Microb., 63, 314-316, 1997;
Such kind of enzyme is already available from Sigma: E.C. 3.4.13.9).

In addition, the koji molds of the invention are particularly adapted for hydrolyzing protein containing materials that comprise at least 5 mM of L-glutamine, allowing formation of L-glutamic acid which is an important natural taste enhancer and
high degree of protein hydrolysates with excellent organoleptic properties.

In a further aspect, the present invention relates to food product comprising a protein hydrolysate obtainable by fermentation of a material comprising proteins and at least 5 mM of L-glutamine with a koji mold of the invention. Such food
contains naturally high amounts of L-glutamic acid (and/or L-glutamate) and high degree of protein hydrolysates with excellent organoleptic properties leading to a non-bitter flavor and a significantly lower allergenicity than unhydrolyzed proteins

Important food product of the present invention is an ingredient of a mother milk substitute for infants, or a hydrolyzed vegetable protein ingredient. The milk substitute may be further formulated in substantially the same way as that indicated
in the prior literature for products of this type (cf. EP 96202475.8).

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention, in addition to those described herein, will become apparent to those skilled in the art from the
foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the claims. Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties to the extent
necessary for understanding the present invention. DNA manipulation, cloning and transformation of bacteria cells are, except where otherwise stated, carried out according to the textbook of Sambrook et al. (Sambrook et al., Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory Press, U.S.A., 1989). These examples are preceded by a brief description of the figures, of the plasmids and strains used, and by the composition of various media. The strains A. oryzae TK3, Aspergilus
oryzae A (see example 1), Aspergilus oryzae NF2 (see example 2), Aspergilus oryzae xprD1 (see example 3) and Aspergilus oryzae NF1 containing pNFF68 (example 4) were deposited under the Budapest Treaty, at the Collection Nationale de Culture de
Microorganismes (CNCM), 25 rue du docteur Roux, 75724 Paris, France, on Jun. 24, 1997, where they receive respectively the deposit numbers CNCM I-1882, CNCM I-1881, CNCM I-1885, CNCM I-1884, CNCM I-1883. All restrictions as to the availability of these
deposits will be withdrawn upon first publication of this application or another application which claims benefit of priority to this application.

FIGURES

FIG. 1 shows the restriction map of pNFF21 which comprises the truncated E. nidulans areA gene and the pkiA promotor and terminater.

FIG. 3 shows the restriction map of the 4.6 kb EcoRI-HindIII insert of plasmid pNFF5, which complements the areA 19 mutation in Emericellia nidulans G332; both exons encompassing the coding region are indicated with solid arrows.

FIG. 4 shows the areA disruption construct pNFF44 containing the two exons of the E. nidulans pyrG gene (pyrl and pyr2), the two exons of A. oryzae areA gene (areA1 and areA2) and the bacterial kanamycin resistance gene (KanaR).

FIG. 5 shows the site directed mutagenesis of the A. oryzae areA gene; the mismatches in the mutagenic primer with the wild type areA sequence are indicted as follows: the stop codon (TAA) is italic, the AfII site doubly underlined and the
introduced EcoRV site is marked in bold print and is underlined.

FIG. 6 shows the relative Endo, LAP and DPPIV activities of A. oryzae TK3 (wild type) and 9 mutants of A. oryzae NF1 which were co-transformed with derepressed areA amplification product and the pyrG amplification product. and transformants were
selected on MM with glucose and glutamine.

Plasmid pFBY182, containing the pepB gene as a EcoRI-XbaI fragment under the control of the Aspergillus niger pkiA promoter and terminator was obtained from Novartis, Switzerland, via Dr. Gabor Jarai (GenBank accession number: S38698).

To assess the feasibility of increasing expression of proteolytic enzymes by modulation of areA expression, we decided to overexpress the Emericellia nidulans gene in A. oryzae TK3.

To this end, amplification of the coding region of the areA gene from Emericellia nidulans G191 and cloning of the PCR product into the expression vector pFBY182 were achieved as follows: with oligonucleotides SEQ ID NO:3 and SEQ ID NO:4, a 2,174
bp fragment, encompassing the areA coding region between positions 2009 and 4168, was amplified from genomic DNA of E. nidulans G191. At the same time an EcoRI site was added to 5' end and a XbaI site to the 3' end, allowing directional cloning into
EcoRI-XbaI digested fungal expression vector pFBY182 to give pNFF21 (see FIG. 1). In pNFF21, areA transcription is under control of the A. niger pkiA promoter and terminator (Graaff, Curr. Genet., 22, 21-27, 1992), thereby preventing the
down-regulation under repressing conditions exerted by its native 3'UTS.

The resulting transformants were screened on MM containing 2% soy protein. Among 20 transformants screened, three showed increased secretion of proteolytic activity as judged from the sizes of the halo surrounding the colony after 36 hours of
incubation at 30.degree. C. (transformants A, B and C). These three transformants were grown for five days at 30.degree. C. in stationary liquid cultures in MM with 0.2% soy protein and analyzed for proteolytic activity with the appropriate controls.

To this end, conidiospores (10.sup.6 /ml) of these three strains were used to inoculate 80 ml of liquid MM with 0.2% soy protein as sole nitrogen and carbon source. These cultures were incubated for 5 days at 30.degree. C. without agitation.
After filtration to remove the mycelium, the medium was assayed for endoproteolytic activity (Endo), Leucine aminopeptidase activity (Lap) and proline-dipeptidyl-peptidase activity (DPPIV). Endoproteolytic enzyme activity was measured with
resorufin-labeled casein according to Boehringer method description supplied with the substrate (Resorufin-labeled casein, Cat.No. 1080733). Leucine aminopeptidase and dipeptidyl peptidase IV activities were determined by UV spectrometry with synthetic
substrates Leu-pNa and Ala-Pro-pNa (Bachem, Switzerland), respectively, according to Sarath et al. (In Protease assay methods for proteolytic enzymes: a practical approach, Beynon R. J., Bond J. S., eds., IRL Press, Oxford). 10 mM substrate stock
solution in dimethylsulfoxide (DMSO) was diluted with 100 mM sodium phosphate buffer, pH 7.0, to a final concentration of 0.5 mM. 20-100 .mu.l culture medium supernatant was added and reaction proceeded for up to 60 min at 37.degree. C. A control with
blank substrate and blank supernatant was assayed in parallel. The release of the chromophoric group 4-nitroaniline (.epsilon.: 10,500 M.sup.-1 cm.sup.-1) was measured at 400 nm and activities were expressed as mU/ml (nmol/min/ml).

Relative proteolytic activities are shown in FIG. 2. In the areA disruption mutant endoproteolytic (Endo) and leucine aminopeptidase (Lap) activity are significantly reduced compared to TK3 and the pyr+ control strains, whereas proline
dipeptidyl peptidase activity (DPPIV) is not affected. Apparently, proline dipeptidylpeptidase expression is not under areA control. Introduction of multiple copies of E. nidulans areA in A. oryzae NF1 under the control of the pkiA expression signals
results in over-expression of endoproteolytic, leucine aminopeptidase and proline-dipeptidyl-peptidase enzyme activity.

EXAMPLE 2

Over-expression of the A. oryzae truncated areA gene

1) Cloning of the A. oryzae areA gene: the A. oryzae areA gene was cloned by complementation of the corresponding areA gene of E. nidulans with the instant library method (Gems et al., 1993).

First of all, the isolation of the genomic DNA was performed according to a modified protocol of the method described by Raeder and Broda (Let. appl. Microbiol., 1, 17-20, 1985). Mycelium was harvested by filtration, immediately frozen in
liquid nitrogen and lyophilized. It was then reduced to a fine powder using a mortar and pestle. 200 mg of the powdered mycelium was resuspended in 2.5 ml of extraction buffer (200 mM Tris-HCl pH 8.5 150 mM NaCl, 25 mM EDTA, 0.5% SDS) and the solution
was extracted with 1.75 ml extraction buffer-equilibrated phenol and 0.75 ml of chloroform/isoamylalcohol (24:1, v/v). The mixture was centrifuged (20 min, 3000 g). The aqueous phase was retrieved and incubated with 125 .mu.l of RNAse A (Boehringer)
solution (10 mg/ml) for 10 min at 37.degree. C. 1.25 ml of 2-propanol (Merck) were then added. The pellet was washed with 70% ethanol and finally resuspended in 500 ml of TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). 500 ml of 2.times.QBT (1.5 M NaCl,
100 mM MOPS, 30% ethanol, pH 7.0) were added to the sample which was then applied to a "Genomic-tip 100" (Qiagen), rinsed and eluted as recommended by the supplier.

The transformation experiment with the partially digested Sau3A A. oryzae TK3 DNA, did not yield any transformants. By contrast the experiments with the BamHI and KpnI digested A. oryzae TK3 DNA did yield 14 and 3 transformants respectively.
Again these transformants exhibited irregular growth, which suggested that the complementing gene was located on an autonomously replicating plasmid. In a separate experiment 40 .mu.g KpnI digested pHELP1 was co-transformed with 100 .mu.g KpnI digested
genomic DNA from E. nidulans G332 (xprD1) and one transformant was obtained.

From three BamHI derived transformants and one KpnI derived area transformant, plasmids were rescued by transformation of E. coli. No plasmids could be isolated from the transformant from the xprD1 transformation. From each individual E.
nidulans BamHI areA.sup.+ transformant several plasmids could be recovered. Restriction analysis of these plasmids showed that they were pHELP1 derivatives containing additional restriction fragments, but that not all of these inserts carried terminal
BamHI sites. Similarly, from the KpnI areA.sup.+ transformant several pHELP1 derivatives could be recovered, non of which had an insert with terminal KpnI sites. These observations indicate instability of the plasmids

One BamHI (pNFF3) and one KpnI (pNFF4) pHELP1 derivative were chosen for further analysis. The inserts of both clones hybridized to the coding region of the E. nidulans areA gene. Detailed analysis of these two clones showed that in pNFF3, the
entire areA gene was located on a 4.6 kb EcoRI-HindIII fragment (FIG. 3). This 4.6 kb EcoRI-HindIII fragment was subcloned into pHSS19 to give pNFF5. Upon re-introduction into E. nidulans G323, pNFF5 restores its ability to grow on NaNO.sub.3 as sole
nitrogen source demonstrating that this plasmid contains a functional areA gene (data not shown).

2) Characterization of the A. oryzae areA gene: the complete nucleotide sequence of the EcoRI-HindIII insert of pNFF5 was determined by analysis of both strands on partially overlapping subclones. The nucleotide sequence was determined, on a
Licor model. 4000 automatic sequencer. IRD41 labeled primers were used for sequencing both strands of partially overlapping subclones by the dideoxynucleotide method of Sanger et al. (Proc Natl Acad Sci USA, 74, 5463-5467, 1977). The DNA sequence
analysis was performed by using the GCG Computer programs (Devereux et al., Nucl. Acids Res., 12, 387-395, 1987).

Results show that the A. oryzae areA gene encodes a protein of 853 amino acid residues with a deduced molecular weight of 91.5 kDa (see SEQ ID NO:2). At the protein level the A. oryzae areA exhibits a similarity of 83% and at the DNA level 70%
similarity with the E. nidulans areA gene.

Moreover, in the putative promoter region the overall DNA homology with E. nidulans drops to 43%. Still, seven stretches of DNA 5 to 13 bp long show 100% sequence identity and occupy virtually identical positions in both promoters. These
sequences could represent cis-acting elements. Additionally, the 5' non-transcribed region contains several putative AREA-binding sites (GATA or TATC; Fu and Marzluf, Proc. Natl. Acad. Sci USA, 87, 5351-5355, 1990) two of which occupy identical
positions as the two functional AREA-binding sites in E. nidulans.

3) Disruption of the A. oryzae areA gene: to elucidate the role of areA in the expression of protease encoding genes, an areA-null mutant was generated by gene disruption. To construct such an areA null allele, the two internal SmaI fragments
(see FIG. 3) were removed from pNFF5 to give pNFF10. To do so, pNFF10 was created by digesting pNFF5, containing the A. oryzae TK3 areA gene, with SmaI and self-ligating the vector containing fragment. This deleted the internal 0.5 and 0.2 kb SmaI
fragments from the second exon of the areA gene in pNFF5.

As selection marker, a PCR product, encompassing the E. nidulans pyrG gene, was inserted into the unique SmaI site of pNFF10 to give pNFF44 (FIG. 4). Accordingly, with oligonucleotides SEQ ID NO:5 and SEQ ID NO: 6 the pyrG gene was amplified
from E. nidulans G332 and the 1.851 bp PCR product cloned into pGEM-T (Promega) to give pNFF38 and pNFF39. The EcoRI fragment, encompassing the pyrG gene was retrieved from pNFF39, blunt ended with T4 DNA polymerase and cloned into the SmaI site of
pNFF10.

This pNFF44 construct, linearized with EcoRI and NheI, was used to transform A. oryzae NF1, and transform ants were selected on osmotically stabilized MM containing glucose and glutamine as carbon and nitrogen source respectively. All pyrG.sup.+
transformants were further checked for their ability to use nitrate and soy protein as sole nitrogen sources. Four pyrG.sub.+ transformants exhibited greatly reduced or no growth on nitrate MM and three did not form a halo when grown for two days on MM
containing 0.2% soy protein as sole nitrogen and carbon source (data not shown). A Southern blot of SmaI digested genomic DNA of these four and six other pyrG.sub.+ transformants was digested with SmaI and probed with the 4.6 kb EcoRI-HindlII insert of
pNFF5. Only in one of the transformants the two internal SmaI fragments of the areA gene were deleted, identifying this transformant as an areA null-mutant. This areA disruption mutant was called NF2.

The areA mutant NF2 was grown for 5 days at 30.degree. C. without agitation in 80 ml of MM with 0.2% soy protein. The areA mutant grew poorly on MM with 0.2% soy protein. Analysis of the culture broth showed a 75% decrease in total
endoproteolytic activity and a 60% decrease in leucine aminopeptidase activity compared to the A. oryzae TK3 (WT) control (FIG. 2). By contrast the proline dipeptidylpeptidase activity in the areA mutant did not significantly differ from the wild type
control (FIG. 2).

4) Construction of a constitutive areA allele : co-transformation experiments with pNFF5, containing the WT areA gene, did not yield co-transformants that overproduced proteolytic enzymes (data not shown). This suggested tight regulation of the
A. oryzae areA gene.

To allow the constitutive expression of proteolytic enzymes (i.e. in the presence of glutamine), truncation of the areA gene was achieved. By site directed mutagenesis, a stop codon (TAA), an AflII and an EcoRV site were introduced into the 4.6
kb EcoRI-HindIII areA fragment, truncating the AREA protein after amino acid residue 752 (see FIG. 5).

To this end, the EcoRI-HindIII insert of pNFF5 was ligated into pALTER1 and introduced into E. coli JM109 to give pNFF49. By superinfection with the helperphage M13KO7, single stranded DNA was generated from pNFF49 which was used in the site
directed mutagenesis procedure with the Altered sites II kit (Promega). Then 0.05 pmol single stranded pNFF49 was annealed to 0.25 pmol Ampicillin repair oligonucleotide SEQ ID NO:7, 0.25 pmol Tetracycline knock-out oligonucleotide SEQ ID NO: 8 and 1.25
pmol areA/xprD1 mutagenic oligonucleotide SEQ ID NO:9, in 20 .mu.l of 20 mM Tris-HCl pH 7.5, 10 mM MgCl.sub.2 and 50 mM NaCl in a Perkin Elmer Thermocycler programmed to heat the annealing mixture to 75.degree. C. for 5 min and then to cool to
45.degree. C. at a rate of 1.degree. C/min. From 45.degree. C. to 20.degree. the cooling rate was increased to 1.5.degree. C/min. Next 3 .mu.l 100 mM Tris-HC1 pH 7.5, 5 mM dNTPs, 10 mM ATP and 20 mM DTT were added. The mixture was incubated for 90
min at 37.degree. C. with 5U T4 DNA polymerase and 1U T4 DNA ligase. A 3 .mu.l aliquot of the reaction mixture was introduced into E. coli ES1301 by electroporation and transformants were selected in 5 ml LB containing 125 .mu.g/ml ampicillin. The
mutagenised plasmids were recovered from ES1301 and introduced into BZ234.

The 3.5 kb EcoRI-EcoRV fragment was further cloned into pBluescript to give pNFF58. To test functionality pNFF58 was introduced into A. oryzae NF2 (see above) and transformants were selected on OFNB containing NaNO.sub.3 as sole nitrogen source. With pNFF58, 1.5 transformants/.mu.g were obtained and with the control pNFF5, 6 transformants/.mu.g. These data prove that pNFF58 still contains a functional areA gene. The pNFF58 transfornants were screened for proteolytic activity on MM with 0.2%
soy protein and MM with 0.2% soy protein and 10 mM glutamine. On 0.2% soy protein several transformants produced bigger halos that the wild type control (A. oryzae TK3) suggesting that overexpression results in enhanced secretion of proteolytic enzymes. Most transformants produced halos on both media, suggesting derepressed expression of proteolytic enzymes (data not shown).

EXAMPLE 3

Construction of protease-overproducing Koji mould strains.

In order to produce potential production koji mold strains, at least one additional copy of the de-repressed areA allele would need to be introduced into the A. oryzae TK3 derivative NF1. For regulatory reasons, this had to be done without
introducing bacterial sequences, especially antibiotic resistance genes. To this end the inserts of pNFF28 and pNFF58 were amplified by PCR with PfuI DNA polymerase and phosphorylated oligonucleotides SEQ ID NO: 10 and SEQ ID NO: I1. The amplification
products were self-ligated and purified. 10 .mu.g of the pNFF58 amplification product and 10 .mu.g of the pNFF28 amplification product were introduced into A. oryzae NF1 and the transformants were selected on osmotically stabilized MM with 50 mM glucose
and 5 mM glutamine. As a control also 10 .mu.g of pNFF28 was introduced. The plasmid pNFF28 yielded 30 transformants/.mu.g, the pNFF28 PCR product 6 transformants/.mu.g and the pNFF28/pNFF58 PCR products 16 transformants/.mu.g.

The potential co-transformants were screened for increased protease activity on MM with 0.2% soy protein and MM with 0.2% soy protein and 10 mM L-glutamine. Twelve transformants produced more proteolytic activity on both media as indicated by
the increased size of the halo they produced. To quantify the overexpression, nine of them were incubated without agitation for 5 days at 30.degree. C. in 80 ml MM containing 0.2% soy protein. The culture media were assayed for proteolytic activity
(FIG. 6).

As with the E. nidulans areA gene under control of the A. niger pkiA expression signals (FIG. 2) all three classes of proteolytic activity tested were increased compared to the A. oryzae TK3 wild type and a pyrG.sup.+ derivative of A. oryzae NF1.

Southern analysis of the protease overproducing strains showed that all co-transformants contain 2 to 4 functionally integrated copies of the de-repressed areA gene.

Comparing the observed levels of protease overproduction and the number of functionally integrated copies of de-repressed areA gene, no clear relation was observed. Transformant xprD1 produces the highest level of proteolytic activity and
contains multiple copies of functionally integrated xprD1. However, transformant xprD12 contains far less copies of functionally integrated xprD1 but produces almost as much activity as transform ant xprD1. Furthermore, the hybridization patterns of
xprD6 and xprD7 are very similar, yet xprD6 overproduces all activities tested 1.5 fold but xprD7 overproduces only proline dipeptidylpeptidase.

EXAMPLE 4

Expression of A. oryzae xprD1 allele with the promoter and terminator of the A. oryzae dppIV gene

Co-transformation experiments of example 2 resulted in strains that had muliple copies of pNFF58 integrated in the genome and that overproduced proteolytic activity 2 to 3 fold when compare to the wild type TK3 strain. By contrast, strains with
one copy of pNFF21 (example 1), where E. nidulans areA is under the control of a strong glycolytic promoter resulted in 6 fold over-expression. These data suggest that the native areA promoter is a weak promoter and that expression of a functional
truncated areA under control of a strong promoter gives better results.

To this end, the dppIV gene of A. oryzae TK3 was amplified by PCR with PfuI DNA polymerase and phosphorylated oligonucleotides SEQ ID NO:12 and SEQ ID NO: 13. The PCR product was then digested with Apal and EcoRV enzymes. The digested
ApaI-EcoRV 4.8 kb fragment was subcloned into pALTER1 (Promega) to give pNFF61. Next pNFF61 was subjected to a site directed mutagenesis according to the protocol of Deng et al. (Anal. Biochem., 200, 81, 1992), using the 5'-phosphorylated mutagenic
oligonucleotides SEQ ID NO: 14 and SEQ ID NO:15 according to the manual with Altered sites II kit (Promega) resulting in pNFF62. Using the polymerase enzyme PfuI and the oligonuclotides SEQ ID NO:16 and SEQ ID NO:17, the xprD1 allele was amplified by
PCR, from pNFF58 containing the A. oryzae xprD1 allele, as a 3.4 kb EcoRI-EcoRV fragment. The 2294 bp xprD1 amplification product was then phosphorylated and cloned into the SmaI digested vector pK19 (Pridmore et al., Gene, 56, 309-312, 1987) to give
pNFF64. Finally the NotI-Ecl136III insert from pNFF64 was inserted into NotI-HpaI pNFF62 creating pNFF68 encompassing the xprD1 allele fused to the dppIV promoter and terminater.

PNFF68 was intoduced into A. oryzae NF1 by co-transformation with pNFF28, and primary transformants were screened for increased proteolytic activity on MM plates containing 0.2% soy protein. Five out of 35 transformants exhibited increased halo
sizes compared to A. oryzae TK3. Among the 5 transformants thus selected, one was deposited under the Budapest Treaty at the CNCM, where it received the deposit number CNCM I-1883.

Co-transformants over-expressing proteolytic enzymes and wild type controls were plated on MM plates containing 0.2% soy protein and 5 mM L-glutamine. All the selected co-transformants still produced a halo in the presence of 5 mM glutamine,
whereas the wild type did not, indicating de-repressed expression of proteolytic activity.

To quantify the over-expression, transformants were incubated without agitation for 5 days at 30.degree. C. in 80 ml MM containing 0.2% soy protein. The culture media were then assayed for proteolytic activity. Results show an overproduction
of proteolytic activity of at least 6 fold when compare to the wild type TK3 strain.

EXAMPLES 5

For preparing a fermented soya sauce, a koji is prepared by mixing an Aspergillus oryzae CNCM I-1883 koji culture with a mixture of cooked soya and roasted wheat, the koji is then hydrolyzed in aqueous suspension for 3 to 8 hours at 45.degree.
C. to 60.degree. C. with the enzymes produced during fermentation of the Aspergillus oryzae CNCM I-1 culture, a moromi is further prepared by adding suitable amount of sodium chloride to the hydrolyzed koji suspension, the moromi is left to ferment and
is then pressed and the liquor obtained is pasteurized and clarified.

EXAMPLES 6

For producing a flavoring agent, a aqueous suspension of a mixture of cooked soya and roasted wheat is prepared, the proteins are solubilized by hydrolysis of the suspension with a protease at pH6.0 to 11.0, the suspension is heat-trated at pH
4.6 to 6.5, and the suspension is ripened

with the prolidase enzyme of Sigma and proteolytic enzymes which have been isolated from a liquid medium fermented by Aspergillus oryzae CNCM I-1881.